Human interferon-inducible protein 10 (IP-10) is a member of the .alpha. chemokine family characterized by a CXC motif (cysteines separated by an amino acid) which include platelet factor 4 (PF-4), IL-8, human protoncogene Gro/melanocyte growth-stimulating activity, .beta.-thromboglobulin, neutrophil-activating protein 2, and neutrophil activating protein from epithelial cells (ENA-78). IP-10 was initially described as an immediate early gene induced by IFN-.gamma. in the histiocytic lymphoma cell line U937 (19). Activated human mononuclear cells, keratinocytes, fibroblasts, endothelial cells, and T cells also express the IP-10 gene (19). Both the human IP-10 gene and the presumed murine homologue, crg-2, code for a secreted mature protein with a predicted molecular weight of approximately 8.6 and 8.7 kD, respectively (20,21). Originally thought to be involved in inflammatory processes because of its inducibility of IFN-.gamma. and structural similarity to PF4 and .beta.-thromboglobulin, IP-10 appears to be multifunctional. It inhibited in vitro colony formation by human bone marrow hematopoietic cells (22) and exerted a potent antitumor effect in vivo (23). Recently, IP-10 was reported to be a chemoattractant for human monocytes and activated T lymphocytes, and to promote T cell adhesion to endothelial cells (24).
Angiogenesis, the process of generating new blood vessels leading to neovascularization, is essential during reproduction, embryonic development, tissue and organ growth, and wound healing (1). Unbalanced neovascularization is believed to contribute to the pathogenesis of certain disease states, such as arthritis, psoriasis, hemangiomas, diabetic retinopathy and retrolental fibroplasia, and to allow tumor growth and metastasis to occur (1). Tumor cells must attract new vessels to expand locally and produce metastasis (1,2).
Several compounds have been reported to inhibit endothelial cell proliferation in various experimental systems, including TGF-.beta. (3), thrombospondin (4), IL-1 (5), .gamma. and .alpha. IFN (6), tissue inhibitor of metalloproteinase-1 (TIMP-1) (7), platelet factor 4 (PF4) (8), protamine (9), fumagillin (10) and angiostatin (22).
Fumagillin, a product of Aspergillus fumigatus fresenious (10), AGM-1470, a synthetic homologue of fumagillin (10), thrombospondin (4), a matrix glycoprotein secreted by a variety of cell types, and the recently identified angiostatin, a fragment of plasminogen (25), have all been shown to potently inhibit endothelial cell proliferation in vitro and to suppress angiogenesis in vivo. IFN .alpha. and a fumagillin derivative, AGM-1470, have reached clinical testing as angiogenesis inhibitors (2). IFN-.alpha. has produced beneficial results in the treatment of certain hemangioendotheliomas (26), but the mechanisms by which it acts are poorly understood. A multistep and complex process such as angiogenesis is likely to be under multiple regulatory controls. Inhibition of angiogenesis in vivo by a drug may or may not be the result of a direct inhibition of endothelial cell growth. Curiously, TGF-.beta., a potent inhibitor of endothelial cell proliferation in vitro, does not inhibit, and perhaps stimulates, angiogenesis in vivo (3,15).
One of the first angiogenesis inhibitors to be identified was PF4 (8), a member of the .alpha. chemokine family. In vitro, PF4 is a potent inhibitor of growth factor dependent endothelial cell proliferation (HUVEC), albeit at high (.about.25 .mu.g/ml) concentrations. PF-4 has also been shown to inhibit growth of solid tumors in vivo (U.S. Pat. Nos. 5,086,164 and 5,112,946). In contrast, another member of the .alpha. chemokine family, IL-8, was reported to stimulate umbilical vein endothelial cell proliferation in vitro and to be potently angiogenic in vivo (27,28). Thus, inhibition of angiogenesis is not a property shared among members of any of the recognized subsets of the chemokine superfamily.
Using an experimental athymic mouse model, it has been shown that regression of human Burkitt's lymphoma is induced by intratumor inoculation of Epstein-Barr virus (EBV)-immortalized human B cells (11). Extensive central necrosis associated with endothelial cell damage and intravascular thrombosis often distal to the necrotic tumor tissue is typical of regressing tumors (11). This suggested that tissue ischemia may be central to tumor regression, and raised the possibility that unbalanced angiogenesis might be responsible for regression of Burkitt's lymphoma in this system (11). Analysis of murine cytokine expression showed that IL-6, TNF.alpha., and IP-10, but not other cytokines, are expressed at higher levels by regressing tumors compared to progressing tumors (11). However, the biological functions of IP-10 were not discerned.
The present invention demonstrates the role of IP-10 in regulation of angiogenesis in inflammation and tumor development.